PD-1 polypeptide variant

Abstract

PD-1 polypeptide variants are provided that contain an extracellular domain that specifically binds to PD-L1 and a transmembrane domain or fragment thereof. The present disclosure also provides PD-1Fc fusion proteins that contain an immunoglobulin Fc region and a PD-1 polypeptide variant.

Description

Cross-reference of related applications

[0001] This application claims priority under U.S. Patent No. 63 / 075,641, filed on 8 September 2020, which is incorporated herein by reference in its entirety. Background of the Invention

[0002] Cancer remains one of the leading causes of death worldwide. Recent statistics report that 13% of the world's population dies from cancer. The International Agency for Research on Cancer (IARC) estimates that in 2012, there were 14.1 million new cancer cases and 8.2 million cancer-related deaths worldwide. By 2030, due to population growth and aging, as well as exposure to risk factors such as smoking, unhealthy diet, and lack of exercise, it is projected that the number of cancer cases worldwide will rise to 21.7 million, and cancer deaths to 13 million. Furthermore, the pain and medical costs associated with cancer treatment reduce the quality of life for both cancer patients and their families.

[0003] Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that modulates the immune system response by downregulating the immune system, suppressing T cell inflammatory activity, and promoting self-tolerance. Because PD-1 is overexpressed in cancer, leading to increased T cell depletion and a reduced antitumor response, blocking the PD-1 / PD-L1 inhibitory pathway and enhancing T cell activation has great potential for treating diseases like cancer. [Overview of the project]

[0004] In this application, programmed cell death 1 (PD-1) polypeptide variants are provided, comprising an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein the PD-1 polypeptide variant comprises: an extracellular domain that specifically binds to programmed cell death 1 ligand (PD-L1); and a transmembrane domain or a fragment thereof. In some embodiments, the PD-1 polypeptide variant comprises an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the PD-1 polypeptide variant comprises an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the PD-1 polypeptide variant comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0005] In some embodiments, the PD-1 polypeptide variant includes SEQ ID NO: 4. In some embodiments, the PD-1 polypeptide variant includes SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide variant includes SEQ ID NO: 8.

[0006] The transmembrane domain used in the present invention may be the wild-type transmembrane domain of PD-1 or a fragment thereof, corresponding to amino acid residues 171-191 of SEQ ID NO: 11. In some embodiments, the transmembrane domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues, encompassing all ranges and subranges linked by these values. For example, a transmembrane domain fragment may contain at least 2 amino acid residues, at least 5 amino acid residues, or at least 10 amino acid residues. In some embodiments, the transmembrane domain has 2 amino acid residues corresponding to amino acid residues 171-172 of SEQ ID NO: 11. In some embodiments, the transmembrane domain has 3 amino acid residues corresponding to amino acid residues 171-173 of SEQ ID NO: 11. In some embodiments, the transmembrane domain has 4 amino acid residues corresponding to amino acid residues 171-174 of SEQ ID NO: 11. In some embodiments, the transmembrane domain has five amino acid residues corresponding to amino acid residues 171-175 of SEQ ID NO: 11.

[0007] The definition of a transmembrane domain or fragment includes variants of the wild-type transmembrane domain of PD-1 corresponding to amino acid residues 171-191 of SEQ ID NO: 11, which are modified by addition, deletion, or substitution of 1 to 5 amino acids, including integer values ​​of 1, 2, 3, 4, and 5 amino acids, which may be modified, added, or substituted from the wild-type transmembrane domain of PD-1 corresponding to amino acid residues 171-191 of SEQ ID NO: 11.

[0008] In this application, programmed cell death 1 (PD-1) polypeptide mutants are provided, which include an amino acid sequence having 95% or more sequence identity with residues 24-172 of SEQ ID NO: 11, wherein the mutant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0009] In some embodiments, the polypeptide variant has an enhanced binding affinity to the PD-L1 molecule as compared to the wild-type PD-1 polypeptide. In some embodiments, the polypeptide variant has a binding affinity (K -8 ~1×10 -10 ) for the PD-L1 molecule of about 1×10 D to 1×10 -9 M. In some embodiments, the polypeptide variant has a binding affinity (K -9 ) for the PD-L1 molecule of about 1.10×10 -10 M, about 1.037×10 D M, or about 7.14×10

[0010] Also provided herein is a PD-1Fc fusion protein comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the immunoglobulin Fc region comprises a sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16, or SEQ ID NO: 10 or SEQ ID NO: 16.

[0011] Also provided herein is a PD-1Fc fusion protein comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% sequence identity to residues 24-172 of SEQ ID NO: 11, and the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R

[0012] Also provided in the present application is an Fc-fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and a scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody has an amino acid sequence having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 17 and SEQ ID NO: 18 linked by a peptide bond or a peptide linker sequence.

[0013] Also provided in the present application is an Fc-fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and a scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% sequence identity to residues 24 to 172 of SEQ ID NO: 11, and the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139, and wherein the scFv against the anti-4-1BB antibody has an amino acid sequence having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NO: 17 and SEQ ID NO: 18 linked by a peptide bond or a peptide linker sequence.

[0014] In some embodiments, the immunoglobulin Fc region comprises a sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the scFv against the anti-4-1BB antibody is an amino acid sequence that is at least 70% identical to SEQ ID NO: 22 or SEQ ID NO: 23.

[0015] The BsAb antibody in the present invention has or does not have the effects of reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0016] The BsAb antibody in the present invention is shown to reduce or not reduce hepatotoxicity. In some embodiments, the fusion protein has enhanced binding affinity to the PD-L1 molecule as compared to the wild-type PD-1 polypeptide. In some embodiments, the fusion protein has a binding affinity (K -8 ~1×10 -10 M) for the PD-L1 molecule. In some embodiments, the fusion protein has a binding affinity (K D ) of about 1.10×10 -9 M, about 1.037×10 -9 M, or about 7.14×10 -10 M for the PD-L1 molecule. D

[0017] Also provided herein is a nucleic acid comprising a sequence encoding a PD-1 polypeptide variant, wherein the polypeptide variant comprises a sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. In some embodiments, the nucleic acid further comprises a sequence encoding an immunoglobulin Fc region, wherein the sequence encoding the immunoglobulin Fc region comprises SEQ ID NO: 9.

[0018] Also provided herein is an expression vector comprising any one of the nucleic acids provided herein. In some embodiments, the vector is a viral vector.​

[0019] Furthermore, the present application provides a pharmaceutical composition comprising one of the PD-1 polypeptide variants provided herein or one of the PD-1 fusion proteins provided herein, and a pharmaceutically acceptable carrier.

[0020] The present application also provides a method for treating a subject, comprising the step of treating a disease or condition by administering one of the pharmaceutical compositions provided herein to the subject in need thereof. In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer. [Brief explanation of the drawing]

[0021] [Figure 1] The amino acid sequence of the human PD-1 protein (SEQ ID NO: 11) is shown. [Figure 2] Sequence alignments of wild-type PD-1 (SEQ ID NO: 2), PD-1.m7 (SEQ ID NO: 4), PD-1.m8 (SEQ ID NO: 6), and euPD-1 are shown (SEQ ID NO: 8). [Figure 3] The structure of an exemplary recombinant expression vector for the PD-1 mutant Fc fusion protein is shown. [Figure 4] The results of SDS-PAGE using PD-1 mutant Fc fusion protein, PD-1Fc fusion protein (1), PD-1.m7Fc fusion protein (2), PD-1.m8Fc fusion protein (3), and euPD-1Fc fusion protein (4) are shown. [Figure 5] Figure 5A shows size exclusion chromatography graphs using gel filtration standard, wild-type PD-1Fc fusion protein (Figure 5B), PD-1.m7Fc fusion protein (Figure 5C), PD-1.m8Fc fusion protein (Figure 5D), and euPD-1Fc fusion protein (Figure 5E). [Figure 6] This shows the expression levels of PD-L1 in the PD-L1 high-expression cell line MDA-MB-231 (human breast cancer cells) and the PD-L1 low-expression cell line MCF-7 (human breast cancer cells). [Figure 7] Figure 7A shows the FACS analysis of PD-1Fc cell binding in PD-L1-positive cells. Figure 7B shows the FACS analysis of PD-1Fc cell binding in PD-L1-negative cells. [Figure 8] Figures 8A and 8C show the assay method for Example 7. Figure 8B shows the binding of euPD-1Fc fusion protein, wild-type PD-1Fc fusion protein, and tecentriq to antigen PD-L1. Figure 8D shows the binding of euPD-1Fc fusion protein, wild-type PD-1Fc fusion protein, and tecentriq to antigen PD-L2. [Figure 9] The results of the PD-1 / PD-L1 blockade bioassay in Example 8 are shown. [Figure 10] The control group for the in vivo efficacy study of Example 9 is shown. [Figure 11] The results of tumor size observation for euPD-1Fc and Tecentriq in Example 9 are shown. [Figure 12] The hepatotoxicity index analysis in Example 9 shows the following results: ALT: 17-77 U / L (Figures 12A and 12E), AST: 54-298 U / L (Figures 12B and 12F), BUN: 8-33 mg / dL (Figures 12C and 12G), and T-BIL: 8-33 mg / dL (Figures 12D and 12H). [Figure 13] This shows an Fc-fusion BsAB using euPD-1 and anti-4-1BB antibody as described in Example 10. [Figure 14] The results of SDS-PAGE using Fc-fusion BsAB with euPD-1 and anti-4-1BB antibody as described in Example 10 are shown. [Figure 15]The size exclusion chromatography graph of Fc-fused BsAB using euPD-1 and anti-4-1BB antibody as described in Example 10, obtained by gel filtration standard, is shown. [Figure 16] The SPR results for the euPD-1BsAB construct in Example 10 are shown. [Figure 17] Figure 17A shows the antigen binding assay method used in Example 11. Figure 17B shows the antigen binding results in Example 11. [Figure 18] The results of the 4-1BB / PD-1 combination bioassay in Example 12 are shown. [Modes for carrying out the invention]

[0022] This disclosure describes PD-1 polypeptide variants, which include mutations in the wild-type PD-1 protein that enhance its affinity for the PD-L1 molecule.

[0023] Definition: about The term "about" as used herein in relation to a value refers to a value similar to the referenced value. In general, a person skilled in the art familiar with the context will recognize the degree of relevant variation encompassed by "about" in that context. For example, in some embodiments, the term "about" may include a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% of the referenced value.

[0024] AdministrationAs used herein, the term “administration” typically refers to administering a composition, or a composition, to a subject or system for achieving delivery of a drug contained in a composition. Those skilled in the art will be aware of the various routes that can be used for administration to a subject, e.g., a human, in a suitable environment. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., intratracheal drip infusion), buccal, dermis (e.g., one or more of the following, including topical, intradermal, interdermal, transdermal, etc.), intestinal, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intracerebral, intra-organ (e.g., intrahepatic), mucous membrane, nasal cavity, oral, rectal, subcutaneous, sublingual, topical, trachea (e.g., intratracheal infusion), vagina, vitreous humor, etc. In some embodiments, administration may consist of only a single dose. In some embodiments, administration may involve the application of a fixed dose. In some embodiments, the administration may include intermittent (e.g., multiple doses divided at time intervals) and / or cyclical (e.g., individual doses separated by a common period) administration. In some embodiments, the administration may include continuous administration (e.g., perfusion) over at least a selected period.

[0025] affinity As is known in the art, "affinity" is a measure of the strength with which a particular ligand binds to its partner. Affinity can be measured in various ways. In some embodiments, affinity is measured by quantitative assays. In some of the embodiments, the binding partner concentration can be fixed to an excess ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the binding partner concentration and / or ligand concentration may be varied. In some of the embodiments, affinity can be compared to a reference under equivalent conditions (e.g., concentrations).

[0026] Antibody drugsAs used herein, the term “antibody agent” refers to a drug that specifically binds to a particular antigen. In some embodiments, the term includes any polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and their fragments. In some embodiments, an antibody agent may include one or more sequence elements, such as humanized, primated, or chimeric, as known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more constructs or forms known or developed in the art to leverage the structural and functional characteristics of an antibody in alternative expressions. For example, in embodiments, antibody agents used according to the present invention include: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fd' fragment, Fd fragment, and their isolated CDRs or sets thereof); single-chain Fvs; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); and small modular immunoassays (SMIPs). TMThe format is selected from, but is not limited to, single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies®; Minibodies; BiTE®; Ankyrin Repeat Protein or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; Microproteins; Fynomers®; Centyrins®; and KALBITOR®. In some embodiments, the antibody agent may lack covalent modifications (e.g., glycan attachments) that may occur naturally. In some embodiments, the antibody agent may include covalent modifications (e.g., glycan attachments, payloads [e.g., detectable portion, therapeutic portion, catalytic portion, etc.]) or other pendant groups [e.g., polyethylene glycol, etc.]. In many embodiments, the antibody agent is a polypeptide or comprises such polypeptide, the amino acid sequence of which contains one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, the antibody agent is a polypeptide or comprises such polypeptide, the amino acid sequence of which contains at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) substantially identical to that found in a reference antibody. The polypeptide is included. In some embodiments, the included CDR is substantially identical to the reference CDR in that it has the same sequence as the reference CDR or contains 1 to 5 amino acid substitutions. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.In some embodiments, the included CDR is substantially the same as the reference CDR in that at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR; otherwise, the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR in that 1 to 5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR; otherwise, the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR in that at least one amino acid in the included CDR is substituted compared to the reference CDR; otherwise, the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR in that 1 to 5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR; otherwise, the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the antibody agent is a polypeptide whose amino acid sequence includes a structural element recognized by those skilled in the art as an immunoglobulin variable domain, or comprises such polypeptide. In some embodiments, the antibody agent is a polypeptide protein having a binding domain homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent comprises at least a portion of a chimeric antigen receptor (CAR), or comprises such a chimeric antigen receptor (CAR).

[0027] antigenAs used herein, the term “antigen” refers to a drug that binds to an antibody. In some embodiments, the antigen may or may not bind to an antibody and induce a specific physiological response in an organism. Generally, the antigen may be any chemical entity, such as, for example, small molecules, nucleic acids, polypeptides, carbohydrates, lipids, polymers (including biological polymers [e.g., nucleic acid and / or amino acid polymers] and non-biological polymers [e.g., polymers other than nucleic acid or amino acid polymers]). In some embodiments, the antigen is a polypeptide or comprises a polypeptide. In some embodiments, the antigen is a glycan or comprises a glycan. Those skilled in the art will understand that the antigen may generally be provided in an isolated or pure form, or alternatively, in a crude form (e.g., an extract of the antigen-containing source together with other materials such as a cell extract or other relatively crude preparation). In some specific embodiments, the antigen is present in a cellular context (e.g., the antigen is expressed on the surface of a cell or expressed inside a cell). In some embodiments, the antigen is a recombinant antigen.

[0028] antigen-binding domain As used herein, “antigen-binding domain” refers to an antibody agent or a portion thereof that specifically binds to a target moiety or entity. Typically, the interaction between the antigen-binding domain and its target is non-covalent. In some embodiments, the target moiety or entity may be any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain may be or contain a polypeptide (or a complex thereof). In some embodiments, the antigen-binding domain is part of a fusion polypeptide. In some embodiments, the antigen-binding domain is part of a chimeric antigen receptor (CAR).

[0029] RelatedTwo events or entities are “related” to each other as used herein if they correlate with one entity that is different in existence, level and / or form. For example, a particular entity (e.g., polypeptide, gene signature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder or condition if its existence, level and / or form is associated with the incidence and / or susceptibility of a disease, disorder or condition (e.g., an entire population) to that disease, disorder or condition. In some embodiments, two or more entities are “related” to each other if they interact directly or indirectly to be in physical proximity to each other and / or maintain proximity. In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other; in some embodiments, two or more entities that are physically related to each other are not covalently bonded to each other but are non-covalently bonded, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism and combinations thereof.

[0030] join As used herein, the term “bonding” is generally understood to refer to non-covalent bonds between or between two or more entities. “Direct” bonding includes physical contact between entities or parts; indirect bonding includes physical interaction via physical contact with one or more intermediate entities. Bonding between two or more entities can be evaluated in any of a variety of situations, including when the interacting entities or parts are studied individually or in the context of more complex systems (e.g., covalently or otherwise related to a carrier entity, and / or within a biological system or cell).

[0031] cancerAs used herein, the terms “cancer,” “malignant tumor,” “neoplasm,” “tumor,” and “carcinoma” refer to cells exhibiting a phenotype that expresses abnormal growth characterized by relative and abnormally unregulated / or autonomous growth and a marked loss of regulation of cell proliferation. In some embodiments, a tumor may be or include cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic. This disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the cancers in question may be characterized by solid tumors. In some embodiments, the cancers in question may be characterized by hematological malignancies.

[0032] Generally, examples of different types of cancer known in the field include, for example, hematopoietic carcinomas including leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue carcinomas, squamous cell carcinomas of the mouth, throat, larynx, and lungs; liver cancers; genitourinary cancers; prostate cancers; cervical cancers; bladder cancers; endometrial cancers; renal cell carcinomas; bone cancers; pancreatic cancers; skin cancers; melanomas of the skin or eyes; endocrine cancers; thyroid cancers; parathyroid cancers; head and neck cancers; breast cancers; gastrointestinal cancers and nervous system cancers; benign lesions; papillary adenocarcinomas, etc.

[0033] Chemotherapy drugsAs used herein, the term “chemotherapeutic agent” has the meaning understood in the art to mean one or more cell death promoters, cell growth inhibitors, and / or cytotoxic agents, including, for example, recommended formulations used / not used for the treatment of one or more diseases, disorders, or conditions specifically associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents such as taxanes, maytansine and its analogues), one or more epothyrons, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogues or nucleotide precursor analogues, one or more peptide antibiotics, one or more platinum-based drugs, one or more retinoids, one or more vinca alkaloids, and / or one or more analogues of any of these (i.e., sharing relevant antiproliferative activity). In some specific embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epotilon, etoposide, fluorouracil, gemcitabine, hydroxyurea The chemotherapeutic agents may be one or more of the following, or combinations thereof: idarubicin, imatinib, irinotecan, maytansine and / or analogues (e.g., DM1), mechloretamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexide, teniposide, thioguanine, topotecan, barbicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, the chemotherapeutic agents may be used in conjunction with antibody-drug conjugates.In some embodiments, the chemotherapeutic agent is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-Doxorubicin, Gemtuzumab Ozogamicin, Brentuximab Vedotin, Trastuzumab Emtansine, Inotuzumab Ozogamicin, Glenvatumumab Vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, Anti-PSMA These are found in antibody-drug conjugates selected from the group consisting of ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab fodotin, and lobotuzumab meltansine.

[0034] ModifiedGenerally, the term “modified” refers to a state of alteration by human intervention. For example, a polypeptide is considered “modified” when its polypeptide sequence has been altered by human intervention. For example, in some embodiments of the present invention, a modified polypeptide includes a sequence containing one or more amino acid mutations, deletions, and / or insertions introduced into a reference polypeptide sequence by human intervention. In some embodiments, a modified polypeptide includes a polypeptide that has been fused (i.e., covalently bonded) with one or more additional polypeptides by human intervention to form a fusion polypeptide that does not occur spontaneously in vivo. Similarly, a cell or organism is considered “modified” if it has been altered in such a way that its genetic information is changed (for example, if new genetic material that did not previously exist is introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or if previously existing genetic material is altered or removed, e.g., by substitution or deletion mutations, or by a mating protocol). As is common practice and as understood by those skilled in the art, a modified polypeptide or derivative and / or offspring of a cell is typically still referred to as “modified,” even if the actual alteration was made to the original entity.

[0035] Pharmaceutical composition As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formed in dosage form with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to human or animal subjects. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population.

[0036] Pharmacologically acceptable carriersAs used herein, the term “pharmaceutically acceptable carrier” has the industry-recognized meaning of a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating agent involved in transporting or delivering a useful compound in the present invention into or to a patient in order to perform an intended function. Typically, such a construct is transported or delivered from one organ or part of the body to another organ or part of the body. Each carrier should be “acceptable” in the sense that it is compatible with other components of a formulation containing a useful compound in the present invention and does not harm the patient. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antimicrobial and antifungal agents, absorption retarders, etc., that are compatible with the activity of the useful compound in the present invention and are physiologically acceptable to the patient. The term “pharmaceutically acceptable carrier” may further include pharmaceutically acceptable salts of the useful compound in the present invention. Other additional components that may be included in the pharmaceutical compositions used in the implementation of the present invention are, for example, described in the literature [Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.)] and incorporated by reference herein. A “pharmaceutically acceptable carrier” is generally interchangeable with other components of the composition, is not harmful to the recipient, and is useful in preparing a pharmaceutical composition that is biologically or otherwise undesirable. A “pharmaceutically acceptable carrier” includes one or more carriers. Embodiments include carriers for topical, ocular, parenteral, intravenous, intraperitoneal, intramuscular, sublingual, nasal, or oral administration. A “pharmaceutically acceptable carrier” further includes formulations for preparing aqueous dispersions and sterile powders or dispersions for injection.

[0037] Pharmaceutically acceptable saltsAs used herein, the term “pharmaceutically acceptable salt” has the meaning generally recognized in the art and refers to derivatives of the compounds provided herein, where the parent compound is modified by converting the present acidic or basic moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; etc. pharmaceutically acceptable salts of the compounds provided herein include, for example, conventional non-toxic salts of the parent compound formed from a non-toxic inorganic or organic acid. pharmaceutically acceptable salts of the compounds provided herein can be synthesized by conventional chemical methods from a parent compound containing a basic or acidic moiety.

[0038] Generally, such salts can be prepared by combining the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used. A catalog of suitable salts can be found in the literature [Remington's Pharmaceutical Sciences, 17]. th This is described in [ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977)], and the entire contents of each are incorporated in this application by reference.

[0039] Excipients As used herein, the term “excipient” generally has the meaning recognized in the industry and refers to physiologically compatible additives useful in the preparation of pharmaceutical compositions. Examples of pharmaceutically acceptable carriers and excipients include, for example, the reference [Remington Pharmaceutical Science, 16 th It is listed in the [Ed].

[0040] PolypeptideAs used herein, the term “polypeptide” generally has the art-recognized meaning of a polymer of at least three amino acids. Those skilled in the art will understand that the term “polypeptide” is sufficiently general to include not only polypeptides having the complete sequences cited herein, but also polypeptides exhibiting functional fragments (i.e., fragments possessing at least one activity) of such complete polypeptides. Furthermore, those skilled in the art will understand that protein sequences generally tolerate some substitutions without disrupting activity. Therefore, any polypeptide containing at least three to four and often twenty or more amino acids is included in the term “polypeptide” as used herein, which generally maintains activity with other polypeptides of the same class, shares at least about 30–40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and generally further includes at least one region of higher identity, generally exceeding 90% or 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, proteins may contain native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins may be antibody agents, antibody fragments, their biologically active portions, and / or their characteristic portions.

[0041] RecombinantAs used herein, “recombinant” is intended to mean polypeptides expressed using recombinant expression vectors transfected into polypeptide host cells designed, modified, prepared, expressed, created, manufactured, and / or isolated by recombinant means; polypeptides isolated from recombinant, recombinant combinatorial human polypeptide libraries; polypeptides isolated from animals (e.g., mice, rabbits, sheep, fish, etc.) that are transgenic to or modified to express genes or genes, or one or more of their components(s), parts(s), elements(s), or domains(s), and / or indicate their expression; and / or polypeptides manufactured, expressed, produced, or isolated by any other means involving splicing or ligating selected nucleic acid sequence elements to one another, or chemically synthesizing selected sequence elements and / or producing nucleic acids that encode or indicate one or more of the polypeptide's components(s), parts(s), elements(s), or domains(s). In some embodiments, one or more of such selected sequence elements exist in nature. In some embodiments, one or more such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements are generated from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic sources, such as in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0042] specific bindingAs used herein, the term “specific binding” refers to the ability to identify possible binding partners in the environment in which binding occurs. A binder that interacts with one particular target in the presence of other potential targets is said to “specifically bind” to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binder and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the binder-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of the binder to compete with alternative interactions between its partner and another entity; in some embodiments, specific binding is assessed by performing such detection or determination over a concentration range.

[0043] Subject As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human, including in some embodiments a prenatal human form). In some embodiments, the subject suffers from a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or signs of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or signs of a disease, disorder, or condition. In some embodiments, the subject is a human having one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is being diagnosed and / or has been treated.

[0044] Treatment drugsAs used herein, the term “therapeutic agent” generally refers to any agent that, when administered to an organism, produces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, the suitable population may be a population of model organisms. In some embodiments, the suitable population may be defined by various criteria such as a specific age group, sex, genetic background, or pre-existing clinical condition. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or signs of a disease, disorder, and / or pathological condition. In some embodiments, a “therapeutic agent” is an agent that has been approved or requires approval by a government agency before being marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent that requires a prescription for administration to humans.

[0045] Therapeutic effective amountAs used herein, the term “therapeutically effective dose” means an amount sufficient to treat a disease, disorder, and / or condition when administered to a population that is suffering from or susceptible to the disease, disorder, and / or condition, in accordance with a therapeutic drug regimen. In some embodiments, a therapeutically effective dose is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more of its characteristics, and / or delays its onset. Those skilled in the art will understand that the term “therapeutically effective dose” does not actually require that a successful treatment be achieved in a particular individual. Rather, a therapeutically effective dose may be an amount that, when administered to a patient in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. For example, in some embodiments, the term “therapeutically effective dose” refers to an amount that, when administered to an individual in need of it in the therapeutic context of the present invention, blocks, stabilizes, attenuates, or reverses cancer-supporting processes originating from that individual. In the context of cancer treatment, a “therapeutically effective dose” is the amount administered to an individual diagnosed with cancer that prevents, stabilizes, inhibits, or mitigates further progression of the cancer in the individual. Particularly preferred “therapeutically effective doses” of the compositions described herein (in therapeutic treatment) are useful in reversing the progression of malignant tumors such as pancreatic cancer or in achieving or extending remission of malignant tumors. The therapeutically effective dose administered to an individual to treat the individual’s cancer may be the same as or different from the therapeutically effective dose administered to promote remission or inhibit metastasis. As with most cancer treatments, the treatments described herein should not be interpreted, limited, or restricted as “treatments” for cancer. Rather, the treatment methods relate to the use of the compositions described for “treating” cancer, that is, for bringing about desirable or beneficial changes to the health of an individual with cancer.Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, stabilization of the patient's condition, reduction in tumor size (tumor regression), improvement of vital functions (e.g., improvement of function of cancerous tissue or organ), further reduction or suppression of metastasis, reduction of opportunistic infections, increased survival rate, reduction of pain, improvement of motor function, improvement of cognitive function, improvement of energy levels (vitality, reduction of fatigue), improvement of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Furthermore, (e.g., as a result of the treatment described herein), the regression of a particular tumor in an individual can also be evaluated (e.g., as a result of treatment) by taking a sample of cancer cells from the site of the tumor, such as pancreatic adenocarcinoma, and by testing the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells and verify at the molecular level whether the cancer cells have regressed to a less malignant phenotype. For example, tumor regression induced by the method of the present invention is manifested by a decrease in any of the pro-angiogenic markers discussed above, an increase in anti-angiogenic markers described herein, normalization of metabolic pathways (i.e., a change to a state found in healthy individuals who do not develop cancer), intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer. Those skilled in the art will understand that in some embodiments, a therapeutically effective amount can be formulated and / or administered as a single dose. In some embodiments, a therapeutically effective amount can be formulated and / or administered in multiple doses, for example, as part of a drug regimen.

[0046] mutantAs used herein, in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “mutant” refers to a molecule that exhibits significant structural identity with a reference molecule in the presence or absence of one or more chemical moieties, or at a certain level, but is structurally different from the reference molecule. In some embodiments, a mutant is also functionally different from its reference molecule. Generally, whether a particular molecule is appropriately considered a “mutant” of a reference molecule is determined on the degree of structural identity with the reference molecule. As those skilled in the art will recognize, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a mutant is a distinct molecule that shares one or more such characteristic structural elements but is different from the reference molecule in at least one way. To give just a few examples, a polypeptide may have characteristic sequence elements consisting of a plurality of amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function; a nucleic acid may have characteristic sequence elements consisting of a plurality of nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. In some embodiments, the mutant polypeptide or nucleic acid may differ from the reference polypeptide or nucleic acid as a result of one or more differences in the amino acid or nucleotide sequence. In some embodiments, the mutant polypeptide or nucleic acid exhibits overall sequence identity with the reference polypeptide or nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, the mutant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the mutant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.

[0047] vectorAs used herein, “vector” refers to a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment may be ligated. Another type of vector is a viral vector, which may have an additional DNA segment ligated into a viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can direct the expression of a operably ligated gene. Such vectors are referred to herein as “expression vectors.” Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer’s specifications, or as commonly achieved in the industry, or as described herein. The techniques and procedures described herein can generally be carried out in accordance with conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see the reference [Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)], which is incorporated into this application for any purpose.

[0048] Programmed cell death protein 1 (PD-1) Programmed cell death protein 1 (PD-1) is an inhibitory receptor expressed by all T cells during activation. PD-1 is expressed not only by conventional T cells but also by regulatory T cells, B cells, natural killer (NK) cells, and certain myeloid cell populations. PD-1 modulates the function of effector T cells during a variety of physiological responses, including acute and chronic infections, cancer, autoimmunity, and immune homeostasis. Furthermore, PD-1 often exhibits high and persistent expression levels during antigen encounters, which can occur in chronic infection and cancer environments and can limit protective immunity.

[0049] In the tumor microenvironment, PD-1 and its ligand, programmed cell death ligand 1 (PD-L1), play crucial roles in tumor progression and survival by evading tumor neutralizing immune surveillance. Blocking the PD-1 and PD-L1 inhibitory pathways, thereby enhancing T cell activation, has been shown to yield beneficial antitumor responses and long-term remission in a broad subset of cancer patients. Therefore, the use of inhibitors that block the interaction between PD-L1 and PD-1 (e.g., on T cells) helps prevent PD-1 stimulation, thereby increasing T cell functional signaling and immune cell responses.

[0050] In some embodiments, specific mutations are introduced into wild-type PD-1 to generate PD-1 polypeptide variants, where the specific mutation induces preferential binding to PD-L1. In some embodiments, the PD-1 polypeptide variant is a soluble PD-1 protein. In some embodiments, the PD-1 polypeptide variant includes an extracellular domain and a transmembrane domain or fragment thereof. The transmembrane domain or fragment thereof is described above in this application.

[0051] In some embodiments, the PD-1 polypeptide variant includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. Here, the PD-1 polypeptide variant includes an extracellular domain that specifically binds to PD-L1 and the transmembrane domain or a fragment thereof. In some embodiments, the PD-1 polypeptide variant includes the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0052] In some embodiments, PD-1 polypeptide variants exhibit enhanced binding affinity to the PD-L1 molecule compared to wild-type PD-1 polypeptides. In some embodiments, polypeptide variants bind approximately 1 × 10⁶ times to the PD-L1 molecule. -8 ~1 × 10 -10 M, preferably about 1 × 10 -9 ~1 × 10 -10 Binding affinity of M (K D ) has. In some embodiments, polypeptide variants have approximately 1.10 × 10⁶ of the PD-L1 molecule. -9 M, approx. 1.037×10 -9 M, or approximately 7.14 × 10 -10 Binding affinity of M (K D ) has.

[0053] As used herein, “nucleic acid” is used to include any compound and / or substance containing polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA).

[0054] In some embodiments, the nucleic acid comprises a sequence encoding a PD-1 polypeptide variant, wherein the PD-1 polypeptide variant comprises at least one nucleotide sequence having 95% or more (96% or more, 97% or more, 98% or more, 99% or more) sequence identity with SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0055] [ka] JPEG0007832689000002.jpg163156 JPEG0007832689000003.jpg120155

[0056] In all embodiments described herein, the PD-1 variant may include additional residues at the C-terminus resulting from the selection of restriction endonucleases used for cloning. The additional residues resulting from the cloning selection are preferably limited to one, two, three, or four residues and are given to a dipeptide, preferably an alanine-serine dipeptide. For example, when used as part of a fusion construct, in the case of euPD-1, the sequence of SEQ ID NO: 8 and the C-terminal AS dipeptide are included in the present invention. Such sequences are assumed and included by the level of amino acid sequence identity defined for the PD-1 polypeptide variants of the present invention.

[0057] In some embodiments, the PD-1 polypeptide variant of the present invention comprises a PD-1 variant having 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with residues 24-172 of SEQ ID NO: 11, wherein the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, vectors containing nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variant of this embodiment, and methods for treating a disease or condition (e.g., cancer) in a subject requiring treatment by administering the pharmaceutical composition.

[0058] In the variant of this embodiment, the PD-1 polypeptide variant of the present invention comprises a PD-1 variant having 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with residues 24-172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, a vector containing nucleic acids, a pharmaceutical composition containing the PD-1 polypeptide variant of this embodiment, and a method for treating a disease or condition (e.g., cancer) in a subject requiring treatment by administering the pharmaceutical composition.

[0059] In some embodiments, the PD-1 polypeptide variant of the present invention comprises a PD-1 variant having 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with residues 24-172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, vectors containing nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variant of this embodiment, and methods for treating a disease or condition (e.g., cancer) in a subject requiring treatment by administering the pharmaceutical composition.

[0060] Preferred mutations include D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122V, This includes A125V, A132I, and R139G.

[0061] Fc fusion protein and encoding nucleotide sequence As used herein, “Fc fusion protein” refers to a modified protein consisting of an Fc domain of IgG linked to a polypeptide or protein of interest. Examples of Fc fusion polypeptides or proteins include, but are not limited to, single peptides, ligands activated upon cell surface receptor binding, signaling molecules (e.g., cytokines), extracellular domains of receptors activated upon dimerization, and bait proteins used to identify binding partners in protein microarrays. In some embodiments, the Fc fusion protein acts as an antibody agent that specifically binds to a particular antigen via an antigen-binding domain. In some embodiments, the Fc fusion protein comprises a polypeptide or polypeptide complex containing sufficient immunoglobulin structural elements to confer specific binding.

[0062] Fc is a constant domain selected from IgG1 or its variants, IgG2 or its variants, IgG4 or its variants, and humanized IgG1 / 2 or its variants. Fc plays multiple roles in dimerization for the formation and maintenance of the Y-shaped structure of Ig, and in Fc-mediated effector function and extension of serum half-life. In some embodiments, monomeric Fc has two domains, a second constant domain (CH2) and a third constant domain (CH3). In some embodiments, the fusion protein includes a linker that fuses the Fc domain together with a specific peptide or protein. In some embodiments, the immunoglobulin Fc region is linked to a specific peptide or protein by a peptide bond. In some embodiments, the immunoglobulin Fc region is linked to a specific peptide or protein by a peptide linker sequence. In some embodiments, the linker sequence includes (G4S)2 (SEQ ID NO: 19), (G4S)3 linker (SEQ ID NO: 20), or 218 linker (SEQ ID NO: 21).

[0063] In some embodiments, a specific peptide or protein is ligated to the carboxy-terminus of the immunoglobulin Fc region. In some embodiments, the Fc domain contains a sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) to SEQ ID NO: 10 or SEQ ID NO: 16.

[0064] In some embodiments, a PD-1 polypeptide variant is used to produce a PD-1 fusion protein. In some embodiments, the PD-1 fusion protein may comprise a PD-1 polypeptide variant and an immunoglobulin Fc region. In some embodiments, the PD-1Fc fusion protein comprises an immunoglobulin Fc region and a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more) sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0065] In some embodiments, the immunoglobulin Fc region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 10 or SEQ ID NO: 16.

[0066] In some embodiments, the PD-1Fc fusion protein exhibits enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide. In some embodiments, the PD-1Fc fusion protein binds to the PD-L1 molecule by approximately 1 × 10⁻¹⁶. -8 ~1 × 10 -10 M, preferably about 1 × 10 -9 ~1 × 10 -10 Binding affinity of M (K D ) has. In some embodiments, the PD-1Fc fusion protein is approximately 1.10 × 10¹⁶ relative to the PD-L1 molecule. -9 M, approx. 1.037×10 -9M, or approximately 7.14 × 10 -10 Binding affinity of M (K D ) has.

[0067] As used herein, “nucleic acid” is used to include any compound and / or substance containing polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA).

[0068] In some embodiments, the nucleic acid construct includes an immunoglobulin Fc region and a region encoding a PD-1 polypeptide variant. In some embodiments, the sequence encoding the Fc domain includes a nucleotide sequence having 95% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to SEQ ID NO: 9.

[0069] [ka]

[0070] As previously described, in all embodiments described herein, the PD-1 variant may include additional residues at the C-terminus resulting from the selection of restriction endonucleases used for cloning. The additional residues resulting from cloning selection are preferably limited to one, two, three, or four residues, along with dipeptide addition, preferably an alanine-serine dipeptide. For example, when used as part of a fusion construct, in the case of euPD-1, the sequence of SEQ ID NO: 8 and the C-terminal AS dipeptide are included in the present invention. Such sequences are assumed and included by the level of amino acid sequence identity defined for the PD-1 polypeptide variants of the present invention. Therefore, in the fusion constructs of this embodiment, the residues resulting from cloning may be located before the linker.

[0071] Furthermore, the present invention includes a PD-1Fc fusion protein, in which multiple copies of a PD-1 polypeptide variant are present, each separated by a linker. The term "multiple copies of a PD-1 polypeptide variant" means that there may be two, three, or four PD-1 polypeptide variants, and the variants present may be the same or different. Also, the linkers separating each PD-1 polypeptide variant may be the same or different. In addition, the linkers between PD-1 polypeptide variants may be different from the linkers between the PD-1 polypeptide variant and the polypeptide or protein of interest.

[0072] In some embodiments, the PD-1 polypeptide variant of the PD-1Fc fusion protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to residues 24-172 of SEQ ID NO: 11, wherein the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, vectors containing nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variant of this embodiment, and methods for treating a disease or condition (e.g., cancer) in a subject requiring treatment by administering the pharmaceutical composition.

[0073] In the variant of this embodiment, the PD-1 polypeptide variant of the PD-1Fc fusion protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to residues 24-172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, a vector containing nucleic acids, a pharmaceutical composition containing the PD-1 polypeptide variant of this embodiment, and a method for treating a disease or condition (e.g., cancer) in a subject requiring treatment by administering the pharmaceutical composition.

[0074] In some embodiments, the PD-1 polypeptide variant of the PD-1Fc fusion protein comprises an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to residues 24-172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, a vector containing the nucleic acid, a pharmaceutical composition containing the PD-1 polypeptide variant of this embodiment, and a method for treating a disease or condition (e.g., cancer) in a subject requiring treatment by administering the pharmaceutical composition.

[0075] Preferred mutations are D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122V, This includes A125V, A132I, and R139G.

[0076] As described above, in some embodiments, the immunoglobulin Fc region includes an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with SEQ ID NO: 10 or SEQ ID NO: 16.

[0077] 4-1BB 4-1BB (sometimes referred to as CD137 or TNFRSF9) is a receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily. 4-1BB is commonly expressed in activated T lymphocytes and is a costimulatory molecule involved in immune and autoimmune diseases (references [Kwon et al. PNAS 84:2896, 1987; Kwon et al. PNAS 86:1963, 1989; Son et al. Journal of Immunological Methods 286(1-2):187-201, 2004], each of which is incorporated herein by reference in whole). Human 4-1BB is a 255-amino acid protein expressed on the cell surface in monomeric (30 kDa) and dimeric (55 kDa) forms, and trimerizes with the 4-1BB ligand to transmit signals.

[0078] Furthermore, 4-1BB is constitutively expressed, albeit at low levels, in many cells, including Foxp3+ Tregs and dendritic cells (DCs). Activation by numerous agonists, such as cytokines (e.g., IL-2, IL-4), polyclonal activators (e.g., Con A and PHA), cell surface molecules (e.g., anti-CD3, anti-CD28), and Ca2+ induction and PKC activity promoters (e.g., ionomycin, phorbol myristate acetate), further enhances 4-1BB expression.

[0079] Numerous studies on mouse and human T cells have shown that 4-1BB promotes enhanced cell proliferation, survival, and cytokine production. Studies have indicated that some 4-1BB agonist monoclonal antibodies increase the expression of costimulatory molecules, significantly enhancing the cytolytic T lymphocyte response and resulting in antitumor effects in prophylactic and therapeutic settings. Furthermore, tumor models treated with 4-1BB monotherapy and combination therapy established a durable antitumor-protective T cell memory response. 4-1BB agonists have also been shown to inhibit autoimmune responses in a variety of autoimmune models recognized in the industry. Such dual activity of 4-1BB presents the potential to provide antitumor activity while mitigating the autoimmune side effects associated with immunotherapeutic approaches.

[0080] In some embodiments of this application, the fusion protein may include an anti-4-1BB antibody domain as an immunoglobulin Fc region. Specifically, the anti-4-1BB antibody domain can be produced using a 94kvt clone (International Publication WO2018-127787, whose entire contents are incorporated herein by reference) that possesses an anti-4-1BB antibody domain (94kvt) as a single-chain Fv (scFv). Examples of scFv suitable for anti-4-1BB antibodies include VH-218 linker-VL (HLC218) and VL-218 linker-VH (LHC218). The 94kvt VH sequence is shown in SEQ ID NO: 17, the 94kvt VL sequence is shown in SEQ ID NO: 18, and the 218 linker is shown in SEQ ID NO: 21. Furthermore, the scFv mutant 94kvt HLC 218 is presented in SEQ ID NO: 22, and the scFv mutant 94kvt LHC 218 is presented in SEQ ID NO: 23.

[0081] In some embodiments, the anti-4-1BB antibody domain (94kvt) as a single-stranded Fv (scFv) contains a sequence that is at least 70% identical to (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) of SEQ ID NO: 22 or SEQ ID NO: 23.

[0082] Accordingly, one embodiment of the present invention is an Fc-fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more) sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and an scFv for anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the scFv for anti-4-1BB antibody comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more) sequence identity with the amino acid sequences comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or peptide linker sequence.

[0083] Furthermore, an Fc-fusion BsAb (bispecific antibody) is provided, which includes an immunoglobulin Fc region and a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, where the PD-1 polypeptide variant has an amino acid sequence with more than 95% (e.g., more than 96%, more than 97%, more than 98%, more than 99%) sequence identity with residues 24-172 of SEQ ID NO: 11, and scF against the anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence. The scFv for anti-4-1BB antibody contains an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more) with respect to the amino acid sequence containing SEQ ID NOs. 17 and 18 linked by a peptide bond or peptide linker.

[0084] In some embodiments, the immunoglobulin Fc region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 10 or SEQ ID NO: 16.

[0085] In some embodiments, the scFv for anti-4-1BB antibody has an amino acid sequence that is at least 70% identical to SEQ ID NO: 22 or SEQ ID NO: 23 (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical).

[0086] Examples of linker sequences include the (G4S)2 (sequence number 19), the (G4S)3 linker (sequence number 20), or the 218 linker (sequence number 21).

[0087] The bispecific antibodies described herein have enhanced binding affinity to the PD-L1 molecule and specific binding affinity to 4-1BB compared to wild-type PD-1 polypeptides.

[0088] The BsAb antibody of the present invention has reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effects, or does not have any such effects at all.

[0089] The BsAb antibody of the present invention exhibits reduced or no hepatotoxicity.

[0090] vector In some embodiments, the nucleic acid constructs described above may be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecules may be operably linked to an expression control sequence. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and virus-derived vectors (e.g., any adenovirus (AV), cytomegalovirus (CMV), monkey virus (SV40) vector, adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector). In some embodiments, the expression vector is a viral vector.

[0091] Further sequences can be appended to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is widely known in the industry.

[0092] In some embodiments, the nucleic acid molecule is inserted into a vector capable of expressing a PD-1 polypeptide variant or PD-1Fc fusion protein as described herein, when introduced into appropriate cells.

[0093] Use in treatment In some embodiments, the PD-1 polypeptide variants, PD-1Fc fusion proteins, or nucleic acid constructs described herein may be used to treat subjects in need. In some embodiments, subjects are diagnosed with PD-L1-related disease. In some embodiments, subjects are diagnosed with PD-L1-related cancer. In some embodiments, a pharmaceutical composition comprising a PD-1 polypeptide variant or PD-1Fc fusion protein and a pharmaceutically acceptable carrier may be administered to subjects diagnosed with PD-L1-related disease. In some embodiments, the pharmaceutical composition may be administered in conjunction with one or more additional anti-cancer therapies, including but not limited to ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors.

[0094] The PD-1 / PD-L1 pathway exhibits an adaptive immune resistance mechanism used by tumor cells in response to intrinsic immune antitumor activity. PD-L1 expressed on tumor cells binds to the PD-1 receptor on activated T cells, inhibiting cytotoxic T cells. In some embodiments, PD-1 polypeptide variants are used as PD-L1 inhibitors to treat cancers including, but not limited to, non-small cell lung cancer, lung adenocarcinoma, gastric cancer, and breast cancer. Cancer can refer to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Unregulated cell division and proliferation can form malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer or cancerous tissue may include tumors.

[0095] Cancers suitable for treatment by the methods of this disclosure may include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, cancers for treatment by the methods of this disclosure may include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin and non-Hodgkin lymphomas), blastomas, sarcomas, and leukemias. In some embodiments, cancer may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0096] In some embodiments, the cancer may be an embryonic tumor (Wilms' tumor, hepatoblastoma, rhabdoid, neuroblastoma), a germ cell tumor (yolk sac tumor, immature teratoma, and embryonic carcinoma), a carcinoma (hepatocellular carcinoma and pulmonary squamous cell carcinoma), a sarcoma (malignant rhabdoid tumor and RMS), or a malignant melanoma.

[0097] The fusion protein and bispecific antibody of the present invention have the further advantage of having reduced or no antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effects. Furthermore, the fusion protein and bispecific antibody of the present invention have reduced or no hepatotoxicity.

[0098] In the context of this Specified, all publications, patent applications, patents, and other references referred to herein are, unless otherwise noted, expressly incorporated by reference in their entirety for all purposes as if they were described in their entirety, and their entirety should be considered as part of this disclosure.

[0099] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. In case of any conflict, this specification, including definitions, shall prevail.

[0100] The foregoing description of the present invention provides methods and processes for manufacturing and using the same, so that those skilled in the art can manufacture and use the same, and such possibilities are provided in particular with respect to the subject matter of the appended claims which constitute part of the original specification.

[0101] As used herein, phrases such as "selected from the group consisting of" or "selected from" include mixtures of specific substances.

[0102] Where numerical limits or ranges are explicitly stated in this specification, endpoints are included. Furthermore, all values ​​and subranges within the numerical limits or ranges are included as explicitly stated.

[0103] The foregoing description is provided to enable those skilled in the art to manufacture and use the present invention, and is provided in relation to a specific use and its requirements. Various modifications to preferred embodiments will be apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and uses without departing from the spirit and scope of the invention. Accordingly, the present invention is not intended to be limited to the embodiments presented, but rather should be given the broadest scope consistent with the principles and features disclosed herein.

[0104] Without being limited to the following specific embodiments, the present invention is illustrated by: (1) A programmed cell death 1 (PD-1) polypeptide variant having an amino acid sequence with 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, The aforementioned PD-1 polypeptide variant is: An extracellular domain that specifically binds to programmed cell death ligand 1 (PD-L1); and Transmembrane domain or fragment thereof A programmed cell death ligand (PD-L1) polypeptide variant, including...

[0105] (2) The PD-1 polypeptide variant described in (1), wherein the PD-1 polypeptide variant contains an amino acid sequence having 97% or more sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0106] (3) In (1) or (2), the PD-1 polypeptide variant is a PD-1 polypeptide variant that contains an amino acid sequence having 98% or more sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0107] (4)A PD-1 polypeptide variant as described in any of (1) to (3), wherein the PD-1 polypeptide variant comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0108] (5)(1) The PD-1 polypeptide variant described herein is a PD-1 polypeptide variant containing SEQ ID NO: 4.

[0109] (6)(1) The PD-1 polypeptide variant described herein includes the PD-1 polypeptide variant containing SEQ ID NO: 6.

[0110] (7)(1) The PD-1 polypeptide variant described herein includes the PD-1 polypeptide variant containing SEQ ID NO: 8.

[0111] (8) A PD-1 polypeptide variant as described in any of (1) to (7), wherein the transmembrane domain contains at least two amino acid residues.

[0112] A PD-1 polypeptide variant as described in any of (9)(1) to (8), wherein the transmembrane domain contains at least 5 amino acid residues.

[0113] A PD-1 polypeptide variant as described in any of (10)(1) to (9), wherein the transmembrane domain contains at least 10 amino acid residues.

[0114] (11)(1)~(10), wherein the polypeptide variant has enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide.

[0115] (12) As described in any of (1) to (11), where the polypeptide variant is approximately 1 × 10⁶ relative to the PD-L1 molecule. -8 ~1 × 10 -10 Binding affinity of M (K D A PD-1 polypeptide variant having )

[0116] (13) As described in any of (1) to (12), where the polypeptide variant is approximately 1.10 × 10⁶ relative to the PD-L1 molecule. -9 M, approx. 1.037×10 -9 M, or approximately 7.14 × 10 -10 Binding affinity of M (K D A PD-1 polypeptide variant having )

[0117] (14) Immunoglobulin Fc region; and A PD-1 Fc fusion protein comprising a PD-1 polypeptide variant linked to the carboxy-terminus of an immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0118] A PD-1Fc fusion protein as described in (15)(14), wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 10 or SEQ ID NO: 16.

[0119] A PD-1Fc fusion protein as described in any of (16), (14), or (15), wherein the fusion protein has enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide.

[0120] As described in any of (17), (14), or (16), where the fusion protein is approximately 1 × 10⁶ of the PD-L1 molecule. -8 ~1 × 10 -10 Binding affinity of M (K D A PD-1 Fc fusion protein having )

[0121] In any of (18), (14), or (17), the fusion protein is approximately 1.10 × 10¹⁶ of the PD-L1 molecule. -9 M, approx. 1.037×10 -9 M, or approximately 7.14 × 10 -10 Binding affinity of M (K D A PD-1Fc fusion protein having )

[0122] (19)(14)~(18), wherein the PD-1 polypeptide variant is a PD-1Fc fusion protein in which the carboxy-terminus of the immunoglobulin Fc region is linked by a peptide linker sequence.

[0123] A PD-1Fc fusion protein as described in any of (20)(14)~(19), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0124] A PD-1Fc fusion protein linked by a peptide linker sequence, as described in any of (21), (14), to (20), wherein one or more copies of a PD-1 polypeptide variant are present, wherein the copies may be the same or different.

[0125] A PD-1Fc fusion protein containing two copies of the PD-1 polypeptide variant described in (22)(21).

[0126] A PD-1Fc fusion protein as described in (23), (21), or (22), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0127] (24)(14)~(23) The PD-1Fc fusion protein described herein is selected from the group consisting of SEQ ID NOs. 12, SEQ ID NOs. 13, SEQ ID NOs. 14, and SEQ ID NOs. 15.

[0128] (25) A nucleic acid comprising a sequence encoding a PD-1 polypeptide variant, wherein the polypeptide variant comprises a sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7.

[0129] (26)(25) The nucleic acid further includes a sequence encoding the immunoglobulin Fc region, where the sequence encoding the immunoglobulin Fc region includes sequence number 9.

[0130] (27) An expression vector comprising the nucleic acid described in either (25) or (26) above.

[0131] (28)(27) Here, the vector is a viral vector.

[0132] (29) A PD-1 polypeptide variant as described in any of (1) to (13) or a PD-1 fusion protein as described in any of (14) to (24); and A pharmaceutically acceptable carrier.

[0133] (30) A method for treating a disease or condition in a subject requiring treatment, A method comprising the step of administering the pharmaceutical composition of (29) to a subject to treat a disease or condition.

[0134] The method described in (31)(30), wherein the subject has cancer.

[0135] Methods described in (32)(31), wherein cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0136] A method in which, if the pharmaceutical composition according to any of (33)(30) to (32) contains the PD-1 fusion protein according to any of (14) to (24), a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effect is observed or no effect is observed.

[0137] A method in which, if the pharmaceutical composition described in any of (34)(30) to (33) contains the PD-1 fusion protein described in any of (14) to (24), reduced hepatotoxicity is observed or no hepatotoxicity is observed.

[0138] (35) A programmed cell death 1 (PD-1) polypeptide mutant comprising an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to residues 24-172 of SEQ ID NO: 11, wherein the mutant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0139] The PD-1 polypeptide variants described in (36)(35), wherein the variants include mutations in D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139.

[0140] A PD-1 polypeptide variant as described in (37)(35), wherein the variant includes mutations in D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0141] (38)(35)~(37) The mutations described herein are D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122V, PD-1 polypeptide variants that are A125V, A132I, or R139G.

[0142] A PD-1 polypeptide mutant as described in any of (39), (35), or (38), wherein the PD-1 polypeptide mutant comprises an amino acid sequence having 97% or more sequence identity with respect to residues 24-172 of SEQ ID NO: 11, wherein the mutant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0143] A PD-1 polypeptide mutant as described in any of (40), (35), to (39), wherein the PD-1 polypeptide mutant comprises an amino acid sequence having 98% or more sequence identity with respect to residues 24 to 172 of SEQ ID NO: 11, wherein the mutant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0144] (41)(35)~(40) is a PD-1 polypeptide variant that has enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide.

[0145] As described in any of (42), (35), or (41), where the polypeptide variant is approximately 1 × 10⁶ relative to the PD-L1 molecule. -8 ~1 × 10 -10 Binding affinity (K D A PD-1 polypeptide variant having )

[0146] (43) Immunoglobulin Fc region; and A PD-1Fc fusion protein comprising a PD-1 polypeptide variant described in any of (35) to (42) linked to the carboxy-terminus of an immunoglobulin Fc region by a peptide bond or peptide linker sequence.

[0147] A PD-1Fc fusion protein as described in (44)(43), wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 10 or SEQ ID NO: 16.

[0148] The fusion protein described in (45), (43), or (44) is a PD-1Fc fusion protein having enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide.

[0149] The fusion protein described in any of (46), (43), or (45) is approximately 1 × 10¹⁶ of the PD-L1 molecule. -8 ~1 × 10 -10 Binding affinity of M (K D A PD-1Fc fusion protein having )

[0150] (47)(43)~(46) The PD-1 polypeptide variant, where the PD-1 polypeptide variant is linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide linker sequence, is a PD-1Fc fusion protein.

[0151] A PD-1Fc fusion protein as described in any of (48), (43), or (47), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0152] A PD-1Fc fusion protein as described in any of (49), (43), or (48), wherein there are multiple copies of a PD-1 polypeptide variant, which may be identical or different, and which are linked by a peptide linker sequence.

[0153] A PD-1Fc fusion protein containing two copies of the PD-1 polypeptide variant described in (50)(49).

[0154] A PD-1Fc fusion protein as described in (51), (49), or (50), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0155] A nucleic acid comprising a sequence encoding a PD-1 polypeptide variant as described in any of (52), (35), or (51).

[0156] (53)(52) A nucleic acid further containing a sequence encoding an immunoglobulin Fc region.

[0157] An expression vector comprising the nucleic acid described in (54), (52), or (53).

[0158] (55)(54) Here, the vector is a viral vector.

[0159] (56)(35)~(42) PD-1 polypeptide variant or (43)~(51) PD-1 fusion protein; and Pharmacologically acceptable carriers A pharmaceutical composition containing the following:

[0160] (57) A method for treating a disease or condition in a subject requiring treatment, A method comprising the step of administering the pharmaceutical composition of (56) to a subject to treat a disease or condition.

[0161] The method described in (58)(57), wherein the subject has cancer.

[0162] The method described in (59)(58), wherein cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0163] A method according to any of (60)(57) to (59) in which, if the pharmaceutical composition contains the PD-1 fusion protein according to any of (43) to (51), a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effect is observed or not observed.

[0164] A method according to any one of (61), (57), to (60), wherein a reduction in hepatotoxicity is observed or hepatotoxicity is not observed, in which case the pharmaceutical composition contains the PD-1 fusion protein described in any one of (43), to (51).

[0165] (62) Immunoglobulin Fc region; A PD-1 polypeptide variant linked to the N-terminus of an immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the PD-1 polypeptide variant contains an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and scFv for anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the scFv for anti-4-1BB antibody contains an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to the amino acid sequence containing SEQ ID NOs. 17 and 18 linked by a peptide bond or peptide linker sequence. A bispecific antibody containing this antibody.

[0166] A bispecific antibody as described in (63)(62), wherein the immunoglobulin Fc region contains an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 10 or SEQ ID NO: 16.

[0167] (64)(62) or (63), wherein the PD-1 polypeptide variant is a bispecific antibody linked to the N-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0168] A bispecific antibody as described in any of (65), (62), or (64), wherein the scFv for anti-4-1BB antibody is linked to the C-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0169] A bispecific antibody as described in any of (66), (62), to (65), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0170] A 20-specific antibody as described in any of (67), (62), to (66), wherein the scFv for anti-4-1BB antibody includes SEQ ID NO: 22 or SEQ ID NO: 23, or a SEQ ID NO: 22 or SEQ ID NO: 23 that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical).

[0171] A bispecific antibody as described in any of (68), (62), or (67), wherein the bispecific antibody has enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide and has specific binding affinity to 4-1BB.

[0172] (69)(62)~(68) A bispecific antibody as described in any of these; and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0173] (70) A method for treating a disease or condition in a subject requiring treatment, A method comprising the step of administering the pharmaceutical composition of (69) to a subject to treat a disease or condition.

[0174] The method described in (71)(70), wherein the subject has cancer.

[0175] The method described in (72)(71), wherein cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0176] A method, as described in any of (73)(70) to (72), in which a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effect is observed or not observed.

[0177] A method described in any of (74), (70), to (73), wherein reduced hepatotoxicity is observed or no hepatotoxicity is observed.

[0178] (75) Immunoglobulin Fc region; A PD-1 polypeptide variant linked to the N-terminus of an immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more) sequence identity with residues 24-172 of SEQ ID NO: 11, wherein the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139; and scFv for anti-4-1BB antibody linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the scFv for anti-4-1BB antibody contains an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to the amino acid sequence containing SEQ ID NOs. 17 and 18 linked by a peptide bond or peptide linker sequence. A bispecific antibody containing [specific antibody].

[0179] A bispecific antibody as described in (76)(75), wherein the immunoglobulin Fc region contains an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, or 99% or more) with respect to SEQ ID NO: 10 or SEQ ID NO: 16.

[0180] (77)(75) or (76), a bispecific antibody in which the PD-1 polypeptide variant is linked to the N-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0181] A bispecific antibody as described in any of (78), (75), or (77), wherein the scFv for anti-4-1BB antibody is linked to the C-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0182] A bispecific antibody as described in any of (79), (75), or (78), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0183] A bispecific antibody as described in any of (80), (75), to (79), wherein the scFv for anti-4-1BB antibody contains the sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a sequence that is at least 70% identical to SEQ ID NO: 22 or SEQ ID NO: 23 (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical).

[0184] A bispecific antibody as described in any of (81), (75), or (80), wherein the bispecific antibody has enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide and has specific binding affinity to 4-1BB.

[0185] (82)(75)~(81) Bispecific antibodies as described in any of the above; and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0186] (83) A method for treating a disease or condition in a subject requiring treatment, A method comprising the step of administering the pharmaceutical composition of (82) to a subject to treat a disease or condition.

[0187] The method described in (84)(83), wherein the subject has cancer.

[0188] Methods described in (85)(84), wherein cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0189] A method according to any of (86), (83), to (85), wherein a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effect is observed or not observed.

[0190] A method described in any of (87), (83), to (86), wherein reduced hepatotoxicity is observed or no hepatotoxicity is observed. [Examples]

[0191] The present disclosure will be further illustrated in the following embodiments, which will not limit the scope of the disclosure set forth in the claims.

[0192] Example 1. PD-1 variant PD-1Fc fusion proteins containing PD-1 polypeptide variants were generated using the PD1-G4S linker-Fc_pcDNA3.3 plasmid. The topology of the human PD-1 protein is shown in Table 1. The sequences of human PD-1 and PD-1 polypeptide variants are shown in Figures 1 and 2. Mutations for each PD-1 polypeptide variant are listed in Table 2, where the amino acid residue numbers relate to the full-length human PD-1 wild-type sequence (SEQ ID NO: 11) shown in Figure 1. The PD-1 polypeptide variants were designed to include the extracellular domain (24-170) and a portion of the transmembrane domain (171-172) of the full-length human PD-1 wild-type sequence (SEQ ID NO: 11) shown in Figure 1. Specifically, the PD-1Fc fusion proteins were generated using the animal cell expression vector pcDNA3.3, in which the restriction enzymes EcoRI and BamHI were inserted into the restriction sites. The human PD-1 signal peptide (sequence: Q15116, 1-23) was used as the signal peptide, and the 207aa-230aa IMGT allele IHG1*03 and the 231aa-457aa IMGT allele IHG2*01 were used in the Fc region. The DNA construct encoding the PD-1Fc fusion protein is shown in Figure 3.

[0193] [Table 1]

[0194] [Table 2]

[0195] Example 2. Analysis and characterization of PD-1 polypeptide variants PD-1 polypeptide variants were inserted into plasmids and used to generate PD-1Fc fusion proteins using the Expi293 expression system (Invitrogen). The polypeptide variants were then purified using AktaPure (GE Healthcare) and FibroPrismA columns (GE Healthcare, catalog number 17-0618-01). The purified polypeptide variants were passed through a desalting column (GE Healthcare, catalog number 17-1408-01), and protein concentrations were measured using Multiskan GO (Thermo).

[0196] The results are shown in Table 3. [Table 3]

[0197] Example 3. Analysis of PD-1 polypeptide variants by SDS-PAGE PD-1 polypeptide variants were added to LDS sample buffer (Invitrogen, catalog number B0007), and then a sample reducing agent (Invitrogen, catalog number B0004) was added to the reduction condition group, and the mixture was incubated at 70°C for 10 minutes. SDS running buffer (Bio-rad, catalog number 1610732) was added to the prepared sample, and the sample was run for 30 minutes using Mini-protein TGXStain-Free Gel (Bio-rad, catalog number 456-8096). The results were analyzed using Chemidoc (Bio-rad) (Figure 4).

[0198] Example 4. Analysis of affinity for PD-L1 molecule The affinity of PD-1 polypeptide variants to the PD-L1 molecule was analyzed using surface plasmon resonance (SPR). PD-1 polypeptide variants were diluted to a concentration of 2 ug / mL and immobilized on CM5 tips (GE Healthcare, catalog number BR-1005-30). PD-L1 molecules (Sino, catalog number 10084-H08H) were injected at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 nM with association times of 150 seconds and dissociation times of 240 seconds. The affinity of each PD-1 polypeptide variant was measured and analyzed using a Biacore T200 (GE Healthcare). The results show that the PD-1 polypeptide variant euPD-1 has the highest binding affinity to PD-L1 (Table 4).

[0199] [Table 4]

[0200] Example 5. Size exclusion chromatography PD-1 polypeptide variants were analyzed using HPLC (Agilent Technologies, 1260 infinity II LC system) and a size exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8 × 300 mm, part number 0008541, column number 004E04320E). A gel filtration standard (BIO-RAD, catalog number 151-1901) was used as a control group (Figures 5a-5e).

[0201] Example 6. Cell-binding assay of PD-1 fusion protein We used PD-L1 high-expression cell lines MDA-MB-231 (human breast cancer cells) and PD-L1 low-expression cell lines MCF-7 (human breast cancer cells). Figure 6 shows the expression levels of PD-L1 in each cell line. In Figure 6, it was confirmed that PD-L1 was highly expressed in MDA-MB-231 cells, not MCF7 cells.

[0202] 1.5 × 10 5Individual cells were incubated at 4°C for 20 minutes while being treated with antibodies. The treatment concentrations of euPD-1Fc, PD-1Fc, and Tecentriq (Genetech, atezolizumab, anti-PD-L1 antibody) were serially diluted 2-fold to a total of 12 points at 877.19 nM. After one wash with FACS wash buffer (0.5% FBS + 0.1% NaN3 in DPBS), the cells were treated with anti-hFC-AF488 secondary antibody (Jackson ImmunoResearch) at a rate of 1 μl / well for 20 minutes. After two further washes, FACS analysis was performed.

[0203] FACS analysis confirmed that there was virtually no background signal from the secondary antibody. As shown in Figure 7a, PD-1Fc did not adhere to PD-L1-positive cells within the experimental concentration range, while euPD-1Fc and Tecentriq adhered to PD-L1-positive cells in a dose-dependent manner. As shown in Figure 7b, FACS analysis revealed that none of the three antibodies adhered to PD-L1-negative cells.

[0204] Example 7. PD-1 Fusion Protein Antigen Binding Assay An antigen-binding assay was performed to determine whether euPD-1Fc binds to the antigen PD-L1 or PD-L2.

[0205] The assay was performed as shown in Figures 8a and 8c. Briefly, PD-L1 antigen or PD-L2 antigen was coated onto 96-well immunoplates at a concentration of 1 μg / mL overnight at 4°C, and then treated with 150 μl of 1× assay buffer (Biolegend) for 1 hour to block nonspecific binding. euPD-1Fc, PD-1Fc, and Tecentriq were each treated with 100 μL and incubated for 2 hours. The treatment concentration of the PD-L1 antigen-coated plates was serially diluted 3-fold at 30 μg / mL to a total of 15 points, and the treatment concentration of the PD-L2 antigen-coated plates was serially diluted 3-fold at 100 μg / mL to a total of 12 points. Anti-hFc-HRP (Biolegend) was treated with 100 μl at a concentration of 0.4 μg / mL. After incubation for 1 hour, the reaction was chromogenically treated with TMB and stopped with sulfuric acid after 3 minutes. In all steps except the one after TMB treatment, washing was performed three times with washing buffer, and all steps except the antigen coating step were performed at room temperature. As shown in Figure 8b, the binding of euPD-1Fc and Tecentriq to the antigen PD-L1 was confirmed in a dose-dependent manner. Furthermore, in Figure 8d, only PD-1Fc bound to the antigen PD-L2 in a dose-dependent manner, while euPD-1Fc and Tecentriq did not bind.

[0206] Example 8. PD-1 Fusion Protein Blockade Bioassay A blockade bioassay was performed to confirm the inhibitory effect on the binding of PD-1 and PD-L1 (Bicytogen).

[0207] The bioassay product used (Promega, J1250) expresses luciferase under conditions that inhibit the binding of PD-1 and PD-L1. The bioassay and luciferase assay followed the Promega protocol. Target cells were placed in 4 × 10⁶ wells of a 96-well white plate. 4Cells were seeded at 100 μL / well. After incubation overnight in a 37°C CO2 incubator, antibody and effector cells were treated. The antibodies used were euPD-1Fc, PD-1Fc, Tecentriq, and Keytruda (Merck, pembrolizumab, anti-PD-1 antibody). The treatment concentration was serially diluted 2.5 times at 217.39 nM, for a total of 10 points. After incubation for 6 hours in a 37°C CO2 incubator, cells were treated with luciferin and luciferase assays were performed. As shown in Figure 9a, euPD-1Fc, Tecentriq, and Keytruda showed dose-dependent inhibitory effects, and as shown in Figure 9b, euPD-1Fc showed 2.4 to 4.9 times greater inhibitory effect than PD-1Fc.

[0208] Example 9. Study of the in vivo efficacy of PD-1 fusion protein To validate the efficacy of euPD-1Fc, in vivo tumor growth inhibition studies were conducted. The experiments were performed using female C57BL / 6 mice knocked in with hPD-1.

[0209] As tumor cells, MC38 cells expressing human PD-L1 were used, as shown in Figure 10. Each cell measured 8 × 10⁶ cells. 6 Numerous tumor cells were administered subcutaneously. One week after administration, the size of the tumors was measured, and the mean and standard deviation were approximately the same (i.e., about 100 mm). 3Patients were assigned to treatment groups to ensure a specific outcome. Three antibodies, euPD-1Fc, PD-1Fc, and Tecentriq, were administered intravenously. The excipient DPBS (Gibco) was used as the negative control group. Antibody doses were set at 5 mg / kg and 2 mg / kg based on euPD-1Fc, while for PD-1Fc and Tecentriq at 5 mg / kg, a concentration of 8.77 pM was used, taking molecular weight into consideration. The administration was 100 μl, and five doses were given at 3-day intervals. Tumor size was also measured every 3 or 4 days, and continued until 2 days after the last dose. On the final day of the experiment, blood was collected to check for toxicity, and the concentrations of ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), and T-BIL (total bilirubin) were confirmed using a biochemical analyzer.

[0210] When tumor size was observed, as shown in Figures 11a and 11b, both euPD-1Fc (8.77 μM) and Tecentriq (8.77 μM) inhibited tumor growth compared to the negative control group; however, PD-1Fc (8.77 μM) showed growth comparable to that of the negative control group. Furthermore, as shown in Figures 11c and 11b, euPD-1Fc showed a dose-dependent inhibitory effect, with a 33.9% reduction in tumor size in the low-concentration (2 mg / kg) group compared to the negative control group, and a 66.1% reduction in tumor size in the high-grade (5 mg / kg) group.

[0211] Furthermore, a hepatotoxicity index analysis was performed, which is shown in Figure 12. In the hepatotoxicity index analysis, as shown in Figure 12, all four indicators were within the normal range in the euPD-1Fc administration group (ALT: 17~77 U / L (Figures 12a and 12e), AST: 54~298 U / L (Figures 12b and 12f), BUN: 8~33 mg / dL (Figures 12c and 12g), T-BIL: 8~33 mg / dL (Figures 12d and 12h)), and no hepatotoxicity was observed under the experimental conditions.

[0212] Therefore, it was confirmed that fusion proteins containing euPD-1 and IgG1 variants, as well as bispecific antibodies, do not exhibit hepatotoxicity. Furthermore, the antibodies of the present invention lack ADCC and CDC effects.

[0213] Example 10. Design, fabrication, and characterization of the PD-1 fusion protein euPD-1BsAb. Fc-fusion BsAbs using euPD-1 and anti-4-1BB antibody were designed as shown in Figure 13. A (G4S)2 linker was used to ligate euPD-1 and Fc, and IgG1-modified Fc regions were used for the BsAbs (L234A, L235A, K322A, D356E, L358M).

[0214] A BsAb configuration was also designed, which connects two euPD-1s, using a (G4S)3 linker to connect the two euPD-1s, and was designated euPD-1.2.

[0215] At the C-terminal end, the anti-4-1BB antibody was bound to Fc in the form of scFv; in this case, it was linked with a (G4S)2 linker, and two types of scFv for the anti-4-IBB antibody were used: VH-218 linker-VL (HLC218) and VL-218 linker-VH (LHC218).

[0216] The constructs shown in Table 5 or Figure 13 were prepared. The aforementioned sequences are also provided.

[0217] [Table 5] JPEG0007832689000010.jpg200133 JPEG0007832689000011.jpg172152

[0218] Transient transfection was performed in Expi293F cells to generate euPD-1BsAb (bispecific antibody), which was then purified using a protein A column.

[0219] SDS-PAGE analysis revealed that in the non-reduced state, euPD-1×94kvt HLC218 and euPD-1×94kvt LHC218, each containing one euPD-1 molecule, had a biomass of approximately 160 kDa; in the reduced state, this was observed to be approximately 80 kDa. The euPD-1 doubled forms, euPD-1.2×94kvt HLC218 and euPD-1.2×94kvt LHC218, were observed to have a biomass of approximately 250 kDa; in the reduced state, this was observed to be approximately 125 kDa. Size exclusion chromatography analysis revealed a single peak with a purity of 90% or higher (see Figure 15).

[0220] Surface plasmon resonance (SPR) analysis was performed in the same manner as described in Example 4. Specifically, 25 μg / mL of anti-human Fc antibody was immobilized on a CM5 chip. Then, four types of euPD-1BsAb were flowed at a rate of 10 μg / mL and 10 μl / min for 60 seconds to capture the antibodies, and PD-L1 antigens of 100, 50, 25, 12.5, 3.25, 3.125, and 0 nM were used to analyze the K D Analysis was performed. Figure 16 shows the SPR results for the euPD-1BsAb construct.

[0221] Example 11. Antigen binding assay using euPD-1BsAb construct An antigen-binding assay was performed to determine whether euPD-1BsAbs binds to the antigen PD-L1.

[0222] The assay method was carried out as shown in Fig. 17a. Briefly, after coating a 96-well immunoplate with the PD-L1 antigen at a concentration of 1 μg / mL at 4 °C overnight, 150 μl of 1× assay buffer (Biolegend) was added for 1 hour to block non-specific binding. euPD-1BsAb was added at 100 L and incubated for 2 hours. The treatment concentration was serially diluted 10-fold up to a total of 3 points at 10 μg / mL. 100 μl of biotinylated 4-IBB at a concentration of 1 μg / mL was added for 1 hour. Avidin-HRP (Bioglend) was added at 100 μl at the concentration indicated in the protocol for 30 minutes. After color development with TMB, the reaction was stopped with sulfuric acid after 3 minutes. In all steps except the step after TMB treatment, washing was performed 3 times using washing buffer; all processes except the PD-L1 coating step were carried out at room temperature.

[0223] As a result of the experiment, as shown in Fig. 17b, the binding of euPD-1BsAb to the antigen PD-L1 was confirmed to be dose-dependent.

[0224] Example 12.4-1 BB / PD-1 combination bioassay To confirm the binding inhibition effect between PD-1 and PD-L1 and the 4-1BB activation effect, a bioassay of the 4-1BB / PD-1 combination was performed.

[0225] In this experiment, the bioassay product (Promega, CS1978I10) is an assay system that expresses luciferase when 4-1BB is activated by 4-1BB antibody stimulation and at the same time the interaction between PD-1 / PD-L1 is inhibited. The bioassay and luciferase assay were performed according to the Promega protocol. MDA-MB-231 cells expressing PD-L1 were seeded at 4×10 4Cells were seeded at 100 μL / well. After over-incubation in a 37°C CO2 incubator, the cells were treated with antibodies and PD1+4-1BB effector cells. The antibodies used were euPD-1×94kvt HLC218 and euPD-1×94kvt LHC218. The treatment concentration was 4-fold serial dilutions starting from 60 ng / ml, for a total of 4 points. After culturing for 6 hours in a 37°C CO2 incubator, the cells were treated with luciferin and luciferase assays were performed.

[0226] As shown in Figure 18, the luciferase assay results indicated that euPD-1×94kvt HLC218 and euPD-1×94kvt LHC218 activated 4-1BB and inhibited PD-1 / PD-L1 in a dose-dependent manner.

Claims

1. A programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, An extracellular domain that specifically binds to programmed cell death ligand 1 (PD-L1); and A PD-1 polypeptide variant comprising a transmembrane domain or a fragment thereof, Having a mutation in at least one residue selected from the group consisting of P34, V43, T45, T59, and Q99; It further has a mutation in one or more residues selected from the group consisting of D26, V64, L65, N66, Y68, M70, N74, K78, C93, R114, L122, A125, A132, and R139, The amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 is included. PD-1 polypeptide variant.

2. The PD-1 polypeptide variant according to claim 1, wherein the transmembrane domain comprises at least two amino acid residues.

3. A PD-1 polypeptide variant according to claim 1, having enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide.

4. Approximately 1 × 10⁻¹⁶ molecules per PD-L1 molecule －８ ~1 x 10 －１０ Binding affinity of M (K Ｄ A PD-1 polypeptide variant according to claim 3, having the following characteristics:

5. A programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having 95% or more sequence identity with residues 24-172 of SEQ ID NO: 11, It specifically binds to programmed cell death ligand 1 (PD-L1); Having a mutation in at least one residue selected from the group consisting of P34, V43, T45, T59, and Q99; It further has a mutation in one or more residues selected from the group consisting of D26, V64, L65, N66, Y68, M70, N74, K78, C93, R114, L122, A125, A132, and R139, The mutations are D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122V, A125V, A132I, or R139G. PD-1 polypeptide variant.

6. A PD-1 polypeptide variant according to claim 5, comprising mutations in D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139.

7. A PD-1 polypeptide variant according to claim 5, comprising mutations in D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

8. Immunoglobulin Fc region; and The PD-1 polypeptide variant according to claim 1 or 5 is linked to the carboxyl terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence. PD-1Fc fusion protein.

9. The PD-1Fc fusion protein according to claim 8, wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 10 or SEQ ID NO:

16.

10. The PD-1Fc fusion protein according to claim 8, wherein there are two copies of the PD-1 polypeptide variant, which may be the same or different, and are linked by a peptide linker sequence.

11. The PD-1Fc fusion protein according to claim 8, having enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide.

12. Approximately 1 × 10⁻¹⁶ molecules per PD-L1 molecule －８ ~1 x 10 －１０ Binding affinity of M (K Ｄ The PD-1Fc fusion protein according to claim 11, having )

13. A nucleic acid comprising a sequence encoding a PD-1 polypeptide variant according to claim 1 or 5, wherein the polypeptide variant comprises a sequence having 95% or more sequence identity with SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7.

14. The nucleic acid according to claim 13, further comprising a sequence encoding an immunoglobulin Fc region, wherein the sequence encoding the immunoglobulin Fc region comprises SEQ ID NO:

9.

15. An expression vector comprising the nucleic acid described in claim 13.

16. The vector according to claim 15, which is a viral vector.

17. A PD-1 polypeptide variant according to claim 1 or 5, or a fusion protein comprising the PD-1 polypeptide variant with an immunoglobulin Fc region and the carboxyl terminus of the immunoglobulin Fc region linked by a peptide bond or a peptide linker sequence; and Pharmacologically acceptable carriers A pharmaceutical composition containing the following:

18. A pharmaceutical composition according to claim 17 for the prevention or treatment of cancer.

19. The pharmaceutical composition according to claim 18, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

20. The pharmaceutical composition according to claim 17, wherein, when the pharmaceutical composition contains the fusion protein, a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effect is observed, or no such effect is observed.

21. The pharmaceutical composition according to claim 17, wherein, when the pharmaceutical composition contains the fusion protein, reduced hepatotoxicity is observed or no hepatotoxicity is observed.

22. Immunoglobulin Fc region; A PD-1 polypeptide variant according to claim 1 or 5, which is linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and An scFv for anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or peptide linker sequence, wherein the scFv for anti-4-1BB antibody contains an amino acid sequence having 95% or more sequence identity with the amino acid sequence containing SEQ ID NOs. 17 and 18 linked by a peptide bond or peptide linker sequence. A bispecific antibody containing [specific antibody].

23. The bispecific antibody according to claim 22, wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or more sequence identity with SEQ ID NO: 10 or SEQ ID NO:

16.

24. The bispecific antibody according to claim 22, wherein the PD-1 polypeptide, scFv, or both are linked to the immunoglobulin Fc region by a peptide linker selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO:

21.

25. The bispecific antibody according to claim 22, wherein the scFv for the anti-4-1BB antibody comprises a sequence that is at least 90% identical to SEQ ID NO: 22 or SEQ ID NO:

23.

26. The bispecific antibody according to claim 22, wherein the scFv for the anti-4-1BB antibody includes SEQ ID NO: 22 or SEQ ID NO:

23.

27. The bispecific antibody according to claim 22, having enhanced binding affinity to the PD-L1 molecule compared to the wild-type PD-1 polypeptide, and having specific binding affinity to 4-1BB.

28. The bispecific antibody according to claim 22; and Pharmacologically acceptable carriers A pharmaceutical composition containing the following:

29. The pharmaceutical composition according to claim 28 for the prevention or treatment of cancer.

30. The pharmaceutical composition according to claim 29, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

31. The pharmaceutical composition according to claim 28, wherein a reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) effect is observed, or the effect is not observed.

32. The pharmaceutical composition according to claim 28, wherein reduced hepatotoxicity is observed or no hepatotoxicity is observed.

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